1. Field of the Invention
The present invention relates to a method for detecting nucleic acids by electrochemiluminescence, detector for nucleic acids and method for producing the same. More particularly, the present invention is directed to a method for producing a detector for nucleic acids comprising a DNA chip which is fabricated by immobilizing electrochemiluminescent material such as Ru(bpy)32+ derivatives and probe nucleic acid on a fine gold electrode by self assembly, method for selectively detecting a DNA duplex formed by complementary hybridization of DNA by electrochemiluminescence, and detector for nucleic acids.
2. Description of the Background Art
Generally, methods for detecting nucleic acids such as DNA or RNA are employed in various fields such as biological researches, medical diagnosis, new drug screening, forensic medicine, etc.
Southern blotting is one of those methods, a method for detecting DNA having a specific base sequence. That is, DNA fragments of a test sample are separated by electrophoresis, and the separated DNA fragments are moved onto a solid substrate made of nitrocellulose or nylon membrane, thereby maintaining the relative position of DNA fragments. Next, the DNA fragments fixed on the solid substrate and probe DNA labeled with a radioisotope are hybridized, by which the position of the DNA having a specific base sequence can be learned.
Northern blotting for detecting RNA was also developed by modifying the above method, and the operating principle thereof is not so different from that of the Southern blotting.
However, such a conventional nucleic acid detection method is disadvantageous in that it requires enormous labor forces, time, and resources. To overcome the above problem, DNA chip on which plurality of nucleic acids are located at a known position of the substrate in two dimensional array has been developed.
DNA chip which is formed by arranging DNA fragments of variety of base sequences on the surface of a narrow substrate in high density can be used in finding out the information on DNA of an unknown sample by hybridization between an immobilized DNA and unknown DNA sample complementary thereto. Here, the term xe2x80x9chybridizationxe2x80x9d means that gene subsequences are linked each other to form double-stranded DNA by complementary base pairing of hydrogen bond between the DNA bases of adenine-thymine (Axe2x80x94T), guanine-cytosine (Gxe2x80x94C). Accordingly, it is possible to find out the information of DNA sequences of sample by complementarily hybridizing DNA fragments of sample of interest with DNA probes immobilized on substrate and then detecting labeled DNA probes, or DNA sample or double-stranded DNA formed by hybridization.
The most widely employed labeling method in conventional molecular biology is the radioautography in which target DNA is labeled by radioisotope. As the radioisotope, 32P or 35S or the like is generally used, and the binding state of labeled target DNA and the probe DNA can be detected using photographic films. This method can be easily utilized since it does not require much basic knowledge. However, it is disadvantageous in that, the result cannot be obtained immediately because the detection time takes several hours or even a day, the resolution is in the order of no more than 0.1 through 10 xcexcm. Further, the radioisotope used as a label does not ensure human""s safety.
Therefore, laser-induced fluorescence is widely used recent days. This method has advantages that various luminescent materials are available, and the resolution is fine. In addition, in case of applying a CCD camera to the above method, molecules labeled by luminescent material can be imaged as soon as they are linked with one another, thereby enabling a rapid determination of the result. However, it is disadvantageous in that this method, though most widely used, requires a process for covalently bonding these molecules with luminescent material before measuring DNA sample of interest, and expensive equipments such as a laser, optical measuring attachment, etc. Moreover, as disclosed in U.S. Pat. No. 5,091,652, it also requires a high-priced image scanner in order to scan the two-dimensional surface of the substrate.
Another method used as a nucleic acid detection method is the optical detection method (P. A. Stimpson et al., Pro. Natl. Sci. USA, 91(1995) 6379-6383). The above method is to make evanescent waves using a two-dimensional optical wave guide and light scattering, and to make the waves scattered at a label adsorbed to a DNA capture region on the surface of the wave guide. Particles functioning as the above label are concentrated only at the portion where probe oligomer and DNA fragments are linked, which causes light scattering. In this optical detection method, it is very convenient because the cleaning step is not required. The detection step does not need much time because the hybridization pattern of chips can be directly checked with eyes by a CCD camera or 8mm video camera. However, this method also requires expensive equipments.
In addition, researches on the method for detecting a DNA binding without using a label by surface plasmon resonance (SRP) are recently underway. This method is advantageous in that since changes in refractive index and thickness occurring in the interface between the metallic surface of a thin layer and a solution are detected, DNA binding can be easily detected without using a label. However, the above method has a disadvantage that detection is possible only when approximately 1011 probe DNA is immobilized on the surface of 1 cm2 due to its poor sensitivity.
U.S. Pat. No. 5,312,527 relates to a method for electrochemical detection of DNA hybridization, wherein the results of DNA hybridization is detected using the binding between an electrochemically active metal complex and a double-stranded DNA. Though the manufacturing cost can be reduced, it is disadvantageous in that it has a poor sensitivity.
As described above, the conventional nucleic acid detection methods all have various disadvantages, and thus there has been a need to develop a new method in order to solve these problems. In particular, it is required to develop a method for rapidly detecting a hybridization with DNA with a fine sensitivity without performing a process of covalently bonding a sample with a tracer in advance, and moreover, a small, low-priced detection system for development of a portable diagnostic apparatus.
Accordingly, the present invention provides a low-priced detector for nucleic acids with a high sensitivity by electrochemiluminescence, method for producing the same, diagnostic kit for detecting nucleic acids, and method for detecting nucleic acids.